We have earlier shown that a higher expression of MIF in tumors is associated with poorer survival in PDAC patients. Furthermore, we validated these findings in 3 additional cohorts, including in publicly available data sets. We hypothesized that MIF is a potential therapeutic target in PDAC. To test this hypothesis, we used a genetic strategy by deleting MIF gene in LSL-KrasG12D/+;LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice, a genetically engineered mouse model of PDAC, which mirrors the development, progression, and molecular and histopathological characteristics of human disease. Kaplan-Meier analysis showed that MIF-deficient KPC (MKPC) mice survived significantly longer than that of KPC mice with wild-type MIF . Additionally, MKPC mice showed a significant reduction in metastatic burden. Most of the metastasis occurred in liver of both KPC and MKPC mice. Furthermore, primary tumor cells isolated from MKPC mice showed an enhanced expression of E-cadherin and reduced expression of vimentin, zeb1 and N-cadherin at both the protein and mRNA levels, as compared with primary tumor cells from KPC mice suggesting an attenuation of EMT in MKPC tumors. These findings provided in vivo proof-of-concept that MIF is a potential therapeutic target in PDAC. We are currently using pharmacological MIF inhibitors and anti-MIF antibodies to examine the effect of MIF inhibition on pancreatic cancer growth, progression and survival using several pre-clinical models including KPC mice, PDTX and human pancreatic cancer organoids. Additionally, we assessed the clinical relevance of NOS2/NO signaling in the patients with PDAC and examined the NOS2 expression by qRT-PCR in tumors from 107 early stage, resected cases. Patients were then divided into NOS2-high and NOS2-low groups based on the median value of NOS2 expression. Patients with the tumor NOS2 expression above the median value were defined as NOS2-high group, and the patients with the NOS2 expression lower than the median value constituted NOS2-low group. Kaplan-Meier analysis showed that patients with a higher NOS2 had significantly poorer survival as compared to the patients with lower NOS2 expression in resected tumors (Log-rank test p=0.012). Furthermore, a higher NOS2 in tumors predicted poor prognosis by both univariable (hazard ratio=1.73, 95%CI=1.09-2.75, p=0.020) and multivariable (hazard ratio=1.75, 95%CI=1.10-2.79, p=0.018) Cox-regression analyses. Additionally, immunohistochemical staining showed a significantly higher NOS2 protein expression in tumors as compared to nontumor pancreatic ducts. These findings showed that NOS2 is a candidate prognostic marker in early stage PDAC patients undergoing surgical resection and NOS2/NO signaling may play a role in pancreatic cancer progression. To examine the role of NOS2/NO signaling in pancreatic cancer progression, we used a genetic strategy by deleting the NOS2 gene in KPC mouse model of PDAC and generating NOS2-deficient NKPC mice. Pancreatic tumors in KPC mice expressed a high level of NOS2 protein, which as expected was undetectable in tumors from NKPC mice. Kaplan-Meier analysis showed that NKPC mice survived significantly longer than KPC mice (Log-Rank test, p0.001). Compared to tumors in KPC mice, NKPC tumors showed significantly reduced macrophages infiltration, as determined by immunohistochemical analysis of F4/80, a murine macrophage marker and a marked decrease in the expression of chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP1). Furthermore, qRT/PCR analysis revealed a significant decrease in the expression of mir-21 in NKPC tumors as compared to tumors from KPC mice. These findings, using a genetic strategy, provided in vivo proof-of-concept that targeting NOS2 may have potential therapeutic benefits. Furthermore, NOS2/NO signaling may enhance inflammation and miR-21 expression in PDAC. We are currently pursuing pharmacological inhibition of NOS2 using a small molecule NOS2-specific inhibitor in multiple preclinical models of PDAC to assess the therapeutic effect of NOS2 inhibition on PDAC. Furthermore, based on our observation of a lower expression of miR-21 in pancreatic tumors from NOS2-deficient mice, we are extending our investigation on the mechanistic role of NOS2 signaling by investigating the interactive role of NO and miR-21 in pancreatic cancer progression and disease aggressiveness. Our ongoing research build upon our current findings on the role of MIF and NO in pancreatic cancer. As described, we have recently shown that a higher ( above median) expression of MIF and NOS2 are associated with poor survival in pancreatic cancer. Furthermore, genetic ablation of either MIF or NOS2 in a genetically engineered mouse model of PDAC prolonged survival. We hypothesize that MIF and NOS2 are candidate therapeutic targets in PDAC. Based on the genetic proof-of-concept, as described above that MIF and NOS2 may be potential targets for PDAC, we are pursuing several preclinical studies using KPC mice, patient-derived tumor xenografts and patient organoids. These preclinical studies involve pharmacological inhibition of MIF and NOS2 as single agents or in combination of the standard of care drug gemcitabine to evaluate their effects on the pancreatic cancer growth, progression, and survival. Our major goal is to establish strong preclinical evidence providing key support to initiate a clinical trial at CCR/NCI clinical center. Furthermore, we have extended our investigation on the regulation of MIF- and NO-mediated signaling pathways in pancreatic cancer progression, which includes the interactive role of NO and miR-21 in pancreatic cancer progression, and genetic regulation of MIF activity in disease aggressiveness in PDAC patients. These investigations are being pursued by the generation of NOS2 and mir-21 double knockout mice in the background of pancreas specific p53 and Kras mutation. To examine the genetic regulation of MIF, we have focused on the presence of a higher number of CATT microsatellite repeat in MIF promoter. A higher than 5 CATT repeat enhances the MIF production and severity of several inflammatory diseases. We are testing the hypothesis that a higher than 5 CATT repeats in MIF promoter is associated with poorer survival in pancreatic cancer patients. To test this hypothesis DNA of patients from PLCO cohorts have been examined in collaboration with the DCEG, NCI.